- Email: [email protected]
1,5-O-Dicaffeoyl-quinic Acid as a Novel Potential NMDA Receptor Inhibitor from Traditional Chinese Medicine Database by Virtual Screening
366
Tian X et al. Chinese Herbal Medicines, 2016, 8(4): 366-370 Available online at SciVarse ScienceDirect
Chinese Herbal Medicines (CHM) ISSN 1674-6384
Journal homepage: www.tiprpress.com
E-mail: [email protected]
Original article
1,5-O-Dicaffeoyl-quinic Acid as a Novel Potential NMDA Receptor Inhibitor from Traditional Chinese Medicine Database by Virtual Screening Xing Tian1, 2, Jian Wang3, Jun Zhu3, Yan-hua Fan3, Wei-hong Meng1, Rong Fan1, Qing-chun Zhao1* 1. Department of Pharmacy, General Hospital of Shenyang Military Area Command, Shenyang 110840, China 2. Department of Pharmacy, Shihezi University, Shihezi 832003, China 3. Department of Life Science and Biochemistry, Shenyang Pharmaceutical University, Shenyang 110016, China
ARTICLE INFO Article history Received: April 6, 2016 Revised: May 13, 2016 Accepted: June 20, 2016 Available online: October 8, 2016
ABSTRACT Objective Neurodegenerative diseases, such as ischemia, traumatic injury, Alzheimer’s
disease, and Parkinson’s disease are characterized by neuronal loss and dysfunction. It is known that glutamate-induced toxicity plays an important role in neurodegenerative
diseases. Glutamate toxicity seems to be mediated by excessive influx of Ca2+ into neuronal cells through activation of N-methyl-D-aspartate (NMDA) receptor. To search for potential NMDA receptor inhibitors in traditional Chinese medicine. Methods
A
series of computer methods including drug-likeness evaluation, ADMET tests as well as molecular docking have been used. Results
1,5-O-dicaffeoyl-quinic acid was
identified as NMDA receptor inhibitor by virtual screening. Its neuroprotective activity
DOI: 10.1016/S1674-6384(16)60064-3
was further confirmed by in vitro test. 1,5-O-dicaffeoyl-quinic acid showed strong neuroprotection against NMDA-induced cell injury. Conclusion
1,5-O-Dicaffeoyl-
quinic acid may be regarded as a potential NMDA receptor inhibitor for the prevention and treatment of neurodegenerative disorders.
Key words
1,5-O-dicaffeoyl-quinic acid; glutamate; neurodegenerative diseases; N-methyl-Daspartate receptor; virtual screen
1. Introduction Neurodegenerative diseases, such as ischemia, traumatic injury, Alzheimer’s disease, and Parkinson’s disease are characterized by neuronal loss and dysfunction (Newberg et al, 2014). It is known that neurodegenerative diseases give great burden to family and society. Neurodegeneration is a process involved in a complicated series of molecular and biochemical mechanisms that damage the neurologic functions mediated by *
Corresponding author: Zhao QC
© 2016 published by TIPR Press. All rights reserved.
inflammation, ionic imbalance, glutamate-induced excitotoxicity, etc (Arundine and Tymianski, 2004). Glutamate toxicity seems to be induced by excessive influx of Ca2+ into neuronal cells through activation of N-methyl-D-aspartate (NMDA) receptors (Wang and Qin, 2010). Further, significant elevation in intracellular Ca2+ triggers several downstream reactions, including nitrosative stress, oxidative stress, and mitochondrial dysfunction, leading to the subsequent initiation of apoptosis (Arundine and Tymianski, 2003). As the Ca 2+
Email: [email protected] Tel/Fax: +86-24-2885 6205
Fund: Key National Science and Technology Specific Project of China (2014ZX09J14101-05C)
Tian X et al. Chinese Herbal Medicines, 2016, 8(4): 366-370 signaling pathway plays an important role during the development and progression of neurodegenerative disorders, reducing excessive Ca2+ production by inhibiting NMDA receptor may be an excellent approach for the treatment of neurodegenerative diseases (Lipton, 2004). In recent years, Chinese materia medica (CMM) has got much attention of the pharmaceutical community, for it is an attractive alternative to conventional Western medicine due to its gentle safety in some cases. Interestingly, there are more than half of important pharmaceutical drugs from natural products and their derivatives (Koehn and Carter, 2005). CMM represents a highly promising resource for discovering bioactive compounds. This study is aimed to search for potential NMDA receptor inhibitors in the treatment of neurodegenerative diseases by computational methods. After filtered by ADMET test, molecular docking and pharmacophore model were finished.
2. Materials and methods
367
module of Discovery Studio 3.0, which is a fast and accurate docking method (Rao et al, 2007). The binding site for the NMDA receptor was defined by co-complexed ligand in the crystal structure. The RMSD value between co-crystallized ligand structure and the predicted pose was evaluated to access the docking accuracy. The docking protocols were set up using the default setting.
2.5 Pharmacophore generation and search NMDA receptor ligands (Mony et al, 2009) with known activities were selected to build pharmacophore. Common ligand features were generated by the HipHop algorithm in Discovery Studio 3.0. The maximum number of pharmacophore models was set to 10. The FAST conformational search option was applied to each CMM candidate. The maximum number of conformers generated was set to 250.
2.6 MTT assay
2.1 Preparing ligands 41491 CMM 3D small molecules were downloaded from TCM Database@Taiwan (http://tcm.cmu.edu.tw/) (Chen, 2011). All compounds were first filtered by Lipinski’s Rule of Five which describes what a drug-like compound is. Then their ionization states of the functional groups under physiological pH conditions were adjusted by using Accelrys Discovery Studio 3.0.
2.2 ADMET tests ADMET properties, including human intestinal absorption, aqueous solubility, blood-brain penetration, CYP2D6 enzyme inhibition, hepatotoxicity, and plasma protein binding (PPB) were tested by DS 3.0. The model predicts blood-brain penetration derived from over 800 compounds that have the ability to enter the CNS after oral administration (Egan and Lauri, 2002). There are four blood brain barrier (BBB) prediction levels: 0 (very high penetrant), 1 (high), 2 (medium), 3 (low), and 4 (undefined). Compounds with BBB levels ≤ 2 were selected as the candidate molecules.
2.3 Preparing proteins The crystal structure of the NMDA receptor (3QEL) (Karakas et al, 2011) was obtained from RCSB Protein Data Bank (http://www.pdb.org/). The protein was prepared with the Biopolymer module of Sybyl X, including addition of hydrogen atoms, removal of water molecules, and repair of incomplete side chain amides and side chain bumps. CHARMM force field was applied to the prepared protein to minimize energy.
2.4 Molecular docking The virtual screening process was performed by Libdock
Human neuroblastoma SH-SY5Y cells were cultured in DMEM supplemented with 10% fetal bovine serum. SH-SY5Y cells were seeded into 96-well plates at a density of 1.0 × 104 cells/well and grown for 24 h. Then cells were treated with 20 and 40 μmol/L of 1,5-O-dicaffeoyl-quinic acid. After incubation for 2 h, the medium was replaced with Mg2+-free Lock’s buffer (154 mmol/L NaCl, 5.6 mmol/L KCl, 3.6 mmol/L NaHCO3, 2.3 mmol/L CaCl2, 5.6 mmol/L glucose, 5 mmol/L HEPES, pH 7.4) containing 1 mmol/L NMDA. After treated for 30 min, cells were returned to the normal culture medium for another 12 h. At the end of these treatments, cells were treated with the MTT solution (final concentration of 0.5 mg/mL). After incubation for 4 h, the medium was slowly removed, and DMSO was added. The absorbance was measured at 490 nm using a microplate reader (ELX 800, Bio-tek, USA).
3. Results and discussion 3.1 Pharmacologically active compounds in CMM In order to find the effective medicines for neurodegenerative diseases, Lipinski’s Rule of Five served to remove a number of compounds, such as highly glycosylated saponins, which are hardly to be absorbed. In this way, 9649 compounds were selected. Furthermore, before reaching the neuronal cells targets, compounds have to pass the BBB. Therefore, considering the silico predicted results, only compounds with BBB levels ≤ 2 indicating middle blood-brain penetration ability were selected. The number of compounds selected after ADMET test was 5639.
3.2 Docking results In order to estimate whether the docking methods were suitable for NMDA receptor, the control ligand known to bind to the protein was re-docked back into the previous
368
Tian X et al. Chinese Herbal Medicines, 2016, 8(4): 366-370
structure of the protein-ligand complex. As root mean squared deviation (RMSD) value between actual and predicted poses for NMDA receptor was within 2 Å (1.5089 Å), the docking methods were relatively good (Hartshorn et al, 2007). The docking score of the control ligand for the NMDA receptor was 135. There were totally 200 compounds identified by docking results that had higher scores than the control ligands. The top five compounds with highest scores were listed in Figure 1. Figure 2 showed the example of 1,5-Odicaffeoyl-quinic acid docking poses with the NMDA receptor. The docking model suggested that 1,5-Odicaffeoyl-quinic acid interacted with Glu235 and Ile133 in the NMDA receptor to form hydrogen bond (HBond). Also, Figure 2 showed that oxygen atom of 1,5-O-dicaffeoylquinic acid served as HBond acceptor to bind with Gln110 of
the NMDA receptor.
3.3 Pharmacophore confirmation To confirm the docking results, pharmacophore models were built. The HipHop algorithm was applied to generating common ligand features, including hydrophobic group, HBond acceptor, HBond donor, and aromatic ring. Figure 3 showed the examples of 1,5-O-dicaffeoyl-quinic acid mapping with the NMDA receptor. The oxygen atom of 1,5-O-dicaffeoyl-quinic acid mapped to the HBond acceptor (Figure 3). The benzene rings on 1,5-O-dicaffeoyl-quinic acid mapped well with the hydrophobic regions. These results suggested that 1,5-Odicaffeoyl-quinic acid yielded good fit with features of the given pharmacophore model.
meso-hannokinol Score: 151
(4E, 6E)-1, 7-bis(4-hydroxyphenyl)hepta-4,6-dien-3-one Score: 146
1, 5-di-O-caffeoylquinic acid Score: 152 Figure 1
lobeline Score: 153 Chemical structures of five identified compounds B
A
Figure 2
Molecular docking models of 1,5-O-dicaffeoyl-quinic acid with NMDA receptor
(A) Bingding mode of 1,5-O-dicaffeoyl-quinic acid in active site of NMDA receptor. The proteins were shown by ribbon, and compounds were shown by stick style. (B) Two-dimensional diagram of interactions between 1,5-O-dicaffeoyl-quinic acid and NMDA receptor. Dashed lines represent hydrogen bond.
Tian X et al. Chinese Herbal Medicines, 2016, 8(4): 366-370
A
B
Figure 3 Pharmacophore mapping results (A) Pharmacophore results of NMDA receptor inhibitors. (B) Pharmacophore results of 1,5-O-dicaffeoyl-quinic acid mapping with NMDA receptor inhibitors. Hydrophobic region (blue sphere), hydrogen bond donor region (magenta sphere), and positive ionizable region (red sphere) were presented.
3.4 Neuroprotection of 1,5‐O‐dicaffeoyl‐quinic acid As 1,5-O-dicaffeoyl-quinic acid showed both good results in molecular docking and pharmacophore, its activity was tested by MTT assay. NMDA-induced cytotoxicity is the common model applied to the investigation of neuroprotective compounds. The SH-SY5Y cells that treated with 1 mmol/L NMDA for 30 min showed a significant decrease in cell viability [(73.45 ± 3.89)%, as compared with the control group]. However, pretreatment with 1,5-O-dicaffeoyl-quinic acid at 20 and 40 μmol/L remarkably increased the cell viability to (86.72 ± 4.40)% (P < 0.01) and (90.45 ± 4.14)% (P < 0.01), respectively. These results indicated that 1,5-O-dicaffeoyl-quinic acid displayed strong neuroprotection against NMDA-induced cell injury. The neuroprotective effects of 1,5-O-dicaffeoyl-quinic acid may be through inhibition of NMDA receptor.
4. Discussion In the present study, potential inhibitors for NMDA receptor, against neurodegenerative diseases were searched in the TCM database by computational methods. In order to screen compounds with good pharmacokinetic and pharmacodynamic properties, drug-likeness was firstly taken into consideration. These drug-like properties including molecular weight, number of hydrogen bond donors and acceptors, intestinal absorption, aqueous solubility, BBB penetration, CYP2D6 enzyme inhibition and hepatotoxicity. All 200 compounds show good BBB permeability ability (BBB value ≤ 2). However, when taking human intestinal absorption, hepatotoxicity, and cytochrome P450D6 enzyme inhibition properties into consideration, not all compounds show the good properties as expected. In addition, as molecules are not ‘frozen status’ and produce the secondary metabolites in human body, how compounds enter human body and change conformations to interact with proteins are unknown. Caffeoylquinic acid derivatives have shown a broad range of biological activities, including antibacterial, antioxidant
369
anticancer, and anti-α-glucosidase activities (Fiamegos et al, 2011; Ooi et al, 2011; Zhao et al, 2014). Previous research has indicated that caffeoylquinic acid derivatives significantly inhibited Aβ42-induced neurotoxicity in SH-SY5Y cells (Miyamae et al, 2012). However, there are no reports on the effects of 1,5-O-dicaffeoyl-quinic acid against the toxicity of NMDA. Present study demonstrates that 1,5-O-dicaffeoylquinic acid may be considered as a new NMDA receptor inhibitor. Further investigations are needed to illustrate the neuroprotection mechanisms.
5. Conclusion In the present study, natural products in TCM database are exploited to discover new compounds with potent efficacy to inhibit the NMDA receptor in neurodegenerative diseases. 1,5-O-dicaffeoyl-quinic acid is identified as NMDA receptor inhibitor by virtual screening. Its neuroprotective activity is further confirmed by in vitro test. In summary, 1,5-Odicaffeoyl-quinic acid may be regarded as a potential NMDA receptor inhibitor for the prevention and treatment of neurodegenerative disorders. However, the underlying mechanisms of neuroprotection should be investigated in future. Conflict of interest statement The authors declare no conflict of interest. References Arundine M, Tymianski M, 2003. Molecular mechanisms of calciumdependent neurodegeneration in excitotoxicity. Cell Calcium 34(4-5): 325-337. Arundine M, Tymianski M, 2004. Molecular mechanisms of glutamate-dependent neurodegeneration in ischemia and traumatic brain injury. Cell Mol Life Sci 61(6): 657-668. Chen CY, 2011. TCM Database@Taiwan: The world’s largest traditional Chinese medicine database for drug screening in silico. PLoS One 6(1): e15939. Egan WJ, Lauri G, 2002. Prediction of intestinal permeability. Adv Drug Deliv Rev 54(3): 273-289. Fiamegos YC, Kastritis PL, Exarchou V, Han H, Bonvin AM, Vervoort J, Lewis K, Hamblin MR, Tegos GP, 2011. Antimicrobial and efflux pump inhibitory activity of caffeoylquinic acids from Artemisia absinthium against gram-positive pathogenic bacteria. PLoS One 6(4): e18127. Hartshorn MJ, Verdonk ML, Chessari G, Brewerton SC, Mooij WT, Mortenson PN, Murray CW, 2007. Diverse, high-quality test set for the validation of protein-ligand docking performance. J Med Chem 50(4): 726-741. Karakas E, Simorowski N, Furukawa H, 2011. Subunit arrangement and phenylethanolamine binding in GluN1/GluN2B NMDA receptors. Nature 475(7355): 249-253. Koehn FE, Carter GT, 2005. The evolving role of natural products in drug discovery. Nat Rev Drug Discov 4(3): 206-220. Lipton SA, 2004. Paradigm shift in NMDA receptor antagonist drug development: molecular mechanism of uncompetitive inhibition by memantine in the treatment of Alzheimer’s disease and other neurologic disorders. J Alzheimers Dis 6(6 Suppl): S61-74. Miyamae Y, Kurisu M, Murakami K, Han J, Isoda H, Irie K,
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Tian X et al. Chinese Herbal Medicines, 2016, 8(4): 366-370
Shigemori H, 2012. Protective effects of caffeoylquinic acids on the aggregation and neurotoxicity of the 42-residue amyloid beta-protein. Bioorg Med Chem 20(19): 5844-5849. Mony L, Krzaczkowski L, Leonetti M, Le Goff A, Alarcon K, Neyton J, Bertrand HO, Acher F, Paoletti P, 2009. Structural basis of NR2B-selective antagonist recognition by N-methyl-D-aspartate receptors. Mol Pharmacol 75(1): 60-74. Newberg AB, Serruya M, Wintering N, Moss AS, Reibel D, Monti DA, 2014. Meditation and neurodegenerative diseases. Ann N Y Acad Sci 1307(1): 112-123. Ooi KL, Muhammad TS, Tan ML, Sulaiman SF, 2011. Cytotoxic, apoptotic and anti-alpha-glucosidase activities of 3,4-di-O-caffeoyl
quinic acid, an antioxidant isolated from the polyphenolic-rich extract of Elephantopus mollis Kunth. J Ethnopharmacol 135(3): 685-695. Rao SN, Head MS, Kulkarni A, LaLonde JM, 2007. Validation studies of the site-directed docking program LibDock. J Chem Inf Model 47(6): 2159-2171. Wang Y, Qin ZH, 2010. Molecular and cellular mechanisms of excitotoxic neuronal death. Apoptosis 15(11): 1382-1402. Zhao JG, Yan QQ, Xue RY, Zhang J, Zhang YQ, 2014. Isolation and identification of colourless caffeoyl compounds in purple sweet potato by HPLC-DAD-ESI/MS and their antioxidant activities. Food Chem 161: 22-26.
Tian X et al. Chinese Herbal Medicines, 2016, 8(4): 366-370 Available online at SciVarse ScienceDirect
Chinese Herbal Medicines (CHM) ISSN 1674-6384
Journal homepage: www.tiprpress.com
E-mail: [email protected]
Original article
1,5-O-Dicaffeoyl-quinic Acid as a Novel Potential NMDA Receptor Inhibitor from Traditional Chinese Medicine Database by Virtual Screening Xing Tian1, 2, Jian Wang3, Jun Zhu3, Yan-hua Fan3, Wei-hong Meng1, Rong Fan1, Qing-chun Zhao1* 1. Department of Pharmacy, General Hospital of Shenyang Military Area Command, Shenyang 110840, China 2. Department of Pharmacy, Shihezi University, Shihezi 832003, China 3. Department of Life Science and Biochemistry, Shenyang Pharmaceutical University, Shenyang 110016, China
ARTICLE INFO Article history Received: April 6, 2016 Revised: May 13, 2016 Accepted: June 20, 2016 Available online: October 8, 2016
ABSTRACT Objective Neurodegenerative diseases, such as ischemia, traumatic injury, Alzheimer’s
disease, and Parkinson’s disease are characterized by neuronal loss and dysfunction. It is known that glutamate-induced toxicity plays an important role in neurodegenerative
diseases. Glutamate toxicity seems to be mediated by excessive influx of Ca2+ into neuronal cells through activation of N-methyl-D-aspartate (NMDA) receptor. To search for potential NMDA receptor inhibitors in traditional Chinese medicine. Methods
A
series of computer methods including drug-likeness evaluation, ADMET tests as well as molecular docking have been used. Results
1,5-O-dicaffeoyl-quinic acid was
identified as NMDA receptor inhibitor by virtual screening. Its neuroprotective activity
DOI: 10.1016/S1674-6384(16)60064-3
was further confirmed by in vitro test. 1,5-O-dicaffeoyl-quinic acid showed strong neuroprotection against NMDA-induced cell injury. Conclusion
1,5-O-Dicaffeoyl-
quinic acid may be regarded as a potential NMDA receptor inhibitor for the prevention and treatment of neurodegenerative disorders.
Key words
1,5-O-dicaffeoyl-quinic acid; glutamate; neurodegenerative diseases; N-methyl-Daspartate receptor; virtual screen
1. Introduction Neurodegenerative diseases, such as ischemia, traumatic injury, Alzheimer’s disease, and Parkinson’s disease are characterized by neuronal loss and dysfunction (Newberg et al, 2014). It is known that neurodegenerative diseases give great burden to family and society. Neurodegeneration is a process involved in a complicated series of molecular and biochemical mechanisms that damage the neurologic functions mediated by *
Corresponding author: Zhao QC
© 2016 published by TIPR Press. All rights reserved.
inflammation, ionic imbalance, glutamate-induced excitotoxicity, etc (Arundine and Tymianski, 2004). Glutamate toxicity seems to be induced by excessive influx of Ca2+ into neuronal cells through activation of N-methyl-D-aspartate (NMDA) receptors (Wang and Qin, 2010). Further, significant elevation in intracellular Ca2+ triggers several downstream reactions, including nitrosative stress, oxidative stress, and mitochondrial dysfunction, leading to the subsequent initiation of apoptosis (Arundine and Tymianski, 2003). As the Ca 2+
Email: [email protected] Tel/Fax: +86-24-2885 6205
Fund: Key National Science and Technology Specific Project of China (2014ZX09J14101-05C)
Tian X et al. Chinese Herbal Medicines, 2016, 8(4): 366-370 signaling pathway plays an important role during the development and progression of neurodegenerative disorders, reducing excessive Ca2+ production by inhibiting NMDA receptor may be an excellent approach for the treatment of neurodegenerative diseases (Lipton, 2004). In recent years, Chinese materia medica (CMM) has got much attention of the pharmaceutical community, for it is an attractive alternative to conventional Western medicine due to its gentle safety in some cases. Interestingly, there are more than half of important pharmaceutical drugs from natural products and their derivatives (Koehn and Carter, 2005). CMM represents a highly promising resource for discovering bioactive compounds. This study is aimed to search for potential NMDA receptor inhibitors in the treatment of neurodegenerative diseases by computational methods. After filtered by ADMET test, molecular docking and pharmacophore model were finished.
2. Materials and methods
367
module of Discovery Studio 3.0, which is a fast and accurate docking method (Rao et al, 2007). The binding site for the NMDA receptor was defined by co-complexed ligand in the crystal structure. The RMSD value between co-crystallized ligand structure and the predicted pose was evaluated to access the docking accuracy. The docking protocols were set up using the default setting.
2.5 Pharmacophore generation and search NMDA receptor ligands (Mony et al, 2009) with known activities were selected to build pharmacophore. Common ligand features were generated by the HipHop algorithm in Discovery Studio 3.0. The maximum number of pharmacophore models was set to 10. The FAST conformational search option was applied to each CMM candidate. The maximum number of conformers generated was set to 250.
2.6 MTT assay
2.1 Preparing ligands 41491 CMM 3D small molecules were downloaded from TCM Database@Taiwan (http://tcm.cmu.edu.tw/) (Chen, 2011). All compounds were first filtered by Lipinski’s Rule of Five which describes what a drug-like compound is. Then their ionization states of the functional groups under physiological pH conditions were adjusted by using Accelrys Discovery Studio 3.0.
2.2 ADMET tests ADMET properties, including human intestinal absorption, aqueous solubility, blood-brain penetration, CYP2D6 enzyme inhibition, hepatotoxicity, and plasma protein binding (PPB) were tested by DS 3.0. The model predicts blood-brain penetration derived from over 800 compounds that have the ability to enter the CNS after oral administration (Egan and Lauri, 2002). There are four blood brain barrier (BBB) prediction levels: 0 (very high penetrant), 1 (high), 2 (medium), 3 (low), and 4 (undefined). Compounds with BBB levels ≤ 2 were selected as the candidate molecules.
2.3 Preparing proteins The crystal structure of the NMDA receptor (3QEL) (Karakas et al, 2011) was obtained from RCSB Protein Data Bank (http://www.pdb.org/). The protein was prepared with the Biopolymer module of Sybyl X, including addition of hydrogen atoms, removal of water molecules, and repair of incomplete side chain amides and side chain bumps. CHARMM force field was applied to the prepared protein to minimize energy.
2.4 Molecular docking The virtual screening process was performed by Libdock
Human neuroblastoma SH-SY5Y cells were cultured in DMEM supplemented with 10% fetal bovine serum. SH-SY5Y cells were seeded into 96-well plates at a density of 1.0 × 104 cells/well and grown for 24 h. Then cells were treated with 20 and 40 μmol/L of 1,5-O-dicaffeoyl-quinic acid. After incubation for 2 h, the medium was replaced with Mg2+-free Lock’s buffer (154 mmol/L NaCl, 5.6 mmol/L KCl, 3.6 mmol/L NaHCO3, 2.3 mmol/L CaCl2, 5.6 mmol/L glucose, 5 mmol/L HEPES, pH 7.4) containing 1 mmol/L NMDA. After treated for 30 min, cells were returned to the normal culture medium for another 12 h. At the end of these treatments, cells were treated with the MTT solution (final concentration of 0.5 mg/mL). After incubation for 4 h, the medium was slowly removed, and DMSO was added. The absorbance was measured at 490 nm using a microplate reader (ELX 800, Bio-tek, USA).
3. Results and discussion 3.1 Pharmacologically active compounds in CMM In order to find the effective medicines for neurodegenerative diseases, Lipinski’s Rule of Five served to remove a number of compounds, such as highly glycosylated saponins, which are hardly to be absorbed. In this way, 9649 compounds were selected. Furthermore, before reaching the neuronal cells targets, compounds have to pass the BBB. Therefore, considering the silico predicted results, only compounds with BBB levels ≤ 2 indicating middle blood-brain penetration ability were selected. The number of compounds selected after ADMET test was 5639.
3.2 Docking results In order to estimate whether the docking methods were suitable for NMDA receptor, the control ligand known to bind to the protein was re-docked back into the previous
368
Tian X et al. Chinese Herbal Medicines, 2016, 8(4): 366-370
structure of the protein-ligand complex. As root mean squared deviation (RMSD) value between actual and predicted poses for NMDA receptor was within 2 Å (1.5089 Å), the docking methods were relatively good (Hartshorn et al, 2007). The docking score of the control ligand for the NMDA receptor was 135. There were totally 200 compounds identified by docking results that had higher scores than the control ligands. The top five compounds with highest scores were listed in Figure 1. Figure 2 showed the example of 1,5-Odicaffeoyl-quinic acid docking poses with the NMDA receptor. The docking model suggested that 1,5-Odicaffeoyl-quinic acid interacted with Glu235 and Ile133 in the NMDA receptor to form hydrogen bond (HBond). Also, Figure 2 showed that oxygen atom of 1,5-O-dicaffeoylquinic acid served as HBond acceptor to bind with Gln110 of
the NMDA receptor.
3.3 Pharmacophore confirmation To confirm the docking results, pharmacophore models were built. The HipHop algorithm was applied to generating common ligand features, including hydrophobic group, HBond acceptor, HBond donor, and aromatic ring. Figure 3 showed the examples of 1,5-O-dicaffeoyl-quinic acid mapping with the NMDA receptor. The oxygen atom of 1,5-O-dicaffeoyl-quinic acid mapped to the HBond acceptor (Figure 3). The benzene rings on 1,5-O-dicaffeoyl-quinic acid mapped well with the hydrophobic regions. These results suggested that 1,5-Odicaffeoyl-quinic acid yielded good fit with features of the given pharmacophore model.
meso-hannokinol Score: 151
(4E, 6E)-1, 7-bis(4-hydroxyphenyl)hepta-4,6-dien-3-one Score: 146
1, 5-di-O-caffeoylquinic acid Score: 152 Figure 1
lobeline Score: 153 Chemical structures of five identified compounds B
A
Figure 2
Molecular docking models of 1,5-O-dicaffeoyl-quinic acid with NMDA receptor
(A) Bingding mode of 1,5-O-dicaffeoyl-quinic acid in active site of NMDA receptor. The proteins were shown by ribbon, and compounds were shown by stick style. (B) Two-dimensional diagram of interactions between 1,5-O-dicaffeoyl-quinic acid and NMDA receptor. Dashed lines represent hydrogen bond.
Tian X et al. Chinese Herbal Medicines, 2016, 8(4): 366-370
A
B
Figure 3 Pharmacophore mapping results (A) Pharmacophore results of NMDA receptor inhibitors. (B) Pharmacophore results of 1,5-O-dicaffeoyl-quinic acid mapping with NMDA receptor inhibitors. Hydrophobic region (blue sphere), hydrogen bond donor region (magenta sphere), and positive ionizable region (red sphere) were presented.
3.4 Neuroprotection of 1,5‐O‐dicaffeoyl‐quinic acid As 1,5-O-dicaffeoyl-quinic acid showed both good results in molecular docking and pharmacophore, its activity was tested by MTT assay. NMDA-induced cytotoxicity is the common model applied to the investigation of neuroprotective compounds. The SH-SY5Y cells that treated with 1 mmol/L NMDA for 30 min showed a significant decrease in cell viability [(73.45 ± 3.89)%, as compared with the control group]. However, pretreatment with 1,5-O-dicaffeoyl-quinic acid at 20 and 40 μmol/L remarkably increased the cell viability to (86.72 ± 4.40)% (P < 0.01) and (90.45 ± 4.14)% (P < 0.01), respectively. These results indicated that 1,5-O-dicaffeoyl-quinic acid displayed strong neuroprotection against NMDA-induced cell injury. The neuroprotective effects of 1,5-O-dicaffeoyl-quinic acid may be through inhibition of NMDA receptor.
4. Discussion In the present study, potential inhibitors for NMDA receptor, against neurodegenerative diseases were searched in the TCM database by computational methods. In order to screen compounds with good pharmacokinetic and pharmacodynamic properties, drug-likeness was firstly taken into consideration. These drug-like properties including molecular weight, number of hydrogen bond donors and acceptors, intestinal absorption, aqueous solubility, BBB penetration, CYP2D6 enzyme inhibition and hepatotoxicity. All 200 compounds show good BBB permeability ability (BBB value ≤ 2). However, when taking human intestinal absorption, hepatotoxicity, and cytochrome P450D6 enzyme inhibition properties into consideration, not all compounds show the good properties as expected. In addition, as molecules are not ‘frozen status’ and produce the secondary metabolites in human body, how compounds enter human body and change conformations to interact with proteins are unknown. Caffeoylquinic acid derivatives have shown a broad range of biological activities, including antibacterial, antioxidant
369
anticancer, and anti-α-glucosidase activities (Fiamegos et al, 2011; Ooi et al, 2011; Zhao et al, 2014). Previous research has indicated that caffeoylquinic acid derivatives significantly inhibited Aβ42-induced neurotoxicity in SH-SY5Y cells (Miyamae et al, 2012). However, there are no reports on the effects of 1,5-O-dicaffeoyl-quinic acid against the toxicity of NMDA. Present study demonstrates that 1,5-O-dicaffeoylquinic acid may be considered as a new NMDA receptor inhibitor. Further investigations are needed to illustrate the neuroprotection mechanisms.
5. Conclusion In the present study, natural products in TCM database are exploited to discover new compounds with potent efficacy to inhibit the NMDA receptor in neurodegenerative diseases. 1,5-O-dicaffeoyl-quinic acid is identified as NMDA receptor inhibitor by virtual screening. Its neuroprotective activity is further confirmed by in vitro test. In summary, 1,5-Odicaffeoyl-quinic acid may be regarded as a potential NMDA receptor inhibitor for the prevention and treatment of neurodegenerative disorders. However, the underlying mechanisms of neuroprotection should be investigated in future. Conflict of interest statement The authors declare no conflict of interest. References Arundine M, Tymianski M, 2003. Molecular mechanisms of calciumdependent neurodegeneration in excitotoxicity. Cell Calcium 34(4-5): 325-337. Arundine M, Tymianski M, 2004. Molecular mechanisms of glutamate-dependent neurodegeneration in ischemia and traumatic brain injury. Cell Mol Life Sci 61(6): 657-668. Chen CY, 2011. TCM Database@Taiwan: The world’s largest traditional Chinese medicine database for drug screening in silico. PLoS One 6(1): e15939. Egan WJ, Lauri G, 2002. Prediction of intestinal permeability. Adv Drug Deliv Rev 54(3): 273-289. Fiamegos YC, Kastritis PL, Exarchou V, Han H, Bonvin AM, Vervoort J, Lewis K, Hamblin MR, Tegos GP, 2011. Antimicrobial and efflux pump inhibitory activity of caffeoylquinic acids from Artemisia absinthium against gram-positive pathogenic bacteria. PLoS One 6(4): e18127. Hartshorn MJ, Verdonk ML, Chessari G, Brewerton SC, Mooij WT, Mortenson PN, Murray CW, 2007. Diverse, high-quality test set for the validation of protein-ligand docking performance. J Med Chem 50(4): 726-741. Karakas E, Simorowski N, Furukawa H, 2011. Subunit arrangement and phenylethanolamine binding in GluN1/GluN2B NMDA receptors. Nature 475(7355): 249-253. Koehn FE, Carter GT, 2005. The evolving role of natural products in drug discovery. Nat Rev Drug Discov 4(3): 206-220. Lipton SA, 2004. Paradigm shift in NMDA receptor antagonist drug development: molecular mechanism of uncompetitive inhibition by memantine in the treatment of Alzheimer’s disease and other neurologic disorders. J Alzheimers Dis 6(6 Suppl): S61-74. Miyamae Y, Kurisu M, Murakami K, Han J, Isoda H, Irie K,
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Tian X et al. Chinese Herbal Medicines, 2016, 8(4): 366-370
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